The goal of this proposal is to establish the functional mechanism of a periplasmic binding protein with respect to its interaction at its membrane receptor site. The arabinose binding protein, of this proposal, is a soluble periplasmic protein capable of recognizing its substrate, L-arabinose, and transferring the substrate unaltered to a membrane integral protein that effects its translocation to the cytoplasm. It is a component of the "high affinity" arabibinos transport system. In this case the source is bacterial, however, the functions are analogous to those of the circulating binding proteins of mammalian systems that recognize and bind small molecules or ions at their point of entry or synthesis and transport them to their specific surface receptors at their site of action. The proposal specifically deals with the identification of the face of the binding protein molecule that interacts with the membrane integral receptor site. One approach will be to use monoclonal antibodies directed against the arabinose binding protein assuming that some subset of such antibodies will interact with the molecule at sites that will prevent the membrane interaction from occurring and not prevent the recognition of substrate. The second approach involves using the existing nucleotide sequence and the tertiary structure of the arabinose binding protein to identify amino acid residues that may be involved in such a membrane interaction and to derive synthetic mutations (by oligonucleotide synthesis and plasmid expression) altering single residues in a defined way. The integrity of the derived mutant protein can be tested by its ability to bind arabinose. By this method the interaction site on the binding protein will be defined in detail. Comparative studies with other binding proteins will be considered. The primary sequence for the membrane integral protein will be derived by determining the nucleotide sequence of the gene ara G. The expression of the gene will be amplified using plasmids and the protein identified and isolated electrophoretically. The regulation of the araF araG operon will be considered under conditions approaching those of the natural environment.